Carbonic anhydrase (CA) is functionally an important enzyme in the central nervous system (CNS) where it is involved in the control of acid-base balance and regulation of the production of cerebrospinal fluid (CSF). Isoenzyme II (CAII) is the most widely distributed CA in the CNS being specifically present in CNS glial tissue and therefore it is expected to be leaked to CSF in degenerative CNS diseases. A competitive dual-labeled time-resolved immunofluorometric assay was developed for simultaneous quantification of human CAI (HCA I) and II (HCA II) in CSF. HCA I was measured to determine the blood contamination in the samples. This solid-phase immunoassay is based on competition between europium (Eu3+)- or samarium (Sm3+)-labeled antigen and the sample antigens for polyclonal rabbit antibodies which are attached to microtiter-plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay, including the separation of free and bound HCA I and II, requires only one incubation step, after which an enhancement solution dissociates Sm3+ and Eu3+ ions from the labeled HCA I and II, respectively, into a solution where they form highly fluorescent chelates. Spectra of the fluorescent chelates in the microtitration strip wells were run on time-resolved fluorometers equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being inversely proportional to the concentration of antigens. The detection limit of the HCA II assay was 0.3 micrograms/l and that of the HCA I assay was 5.2 micrograms/l. The intra- and inter-assay imprecisions (C.V.s) were 8.0% and 8.8% for HCA I and 6.3% and 4.8% for HCA II, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)